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Abstract

Abstract—Hyperhomocysteinemia is a risk factor for atherosclerosis and thrombosis and is inversely related to plasma folate and vitamin B12 levels. We assessed the effects of vitamin supplementation on plasma homocysteine levels in 89 patients with a history of recurrent venous thrombosis and 227 healthy volunteers. Patients and hyperhomocysteinemic (homocysteine level >16 μmol/L) volunteers were randomized to placebo or high-dose multivitamin supplements containing 5 mg folic acid, 0.4 mg hydroxycobalamin, and 50 mg pyridoxine. A subgroup of volunteers without hyperhomocysteinemia was also randomized into three additional regimens of 5 mg folic acid, 0.5 mg folic acid, or 0.4 mg hydroxycobalamin. Before and after the intervention period, blood samples were taken for measurements of homocysteine, folate, cobalamin, and pyridoxal-5′-phosphate levels. Supplementation with high-dose multivitamin preparations normalized plasma homocysteine levels (≤16 μmol/L) in 26 of 30 individuals compared with 7 of 30 in the placebo group. Also in normohomocysteinemic subjects, multivitamin supplementation strongly reduced homocysteine levels (median reduction, 30%; range, −22% to 55%). In this subgroup the effect of folic acid alone was similar to that of multivitamin: median reduction, 26%; range, −2% to 52% for 5 mg folic acid and 25%; range, −54% to 40% for 0.5 mg folic acid. Cobalamin supplementation had only a slight effect on homocysteine lowering (median reduction, 10%; range, −21% to 41%). Our study shows that combined vitamin supplementation reduces homocysteine levels effectively in patients with venous thrombosis and in healthy volunteers, either with or without hyperhomocysteinemia. Even supplementation with 0.5 mg of folic acid led to a substantial reduction of blood homocysteine levels.

Subjects with hyperhomocysteinemia have a twofold to threefold increase in risk of developing cardiovascular disease or venous thrombosis.12345 Reduction of plasma homocysteine levels by vitamins may therefore be of major clinical importance. Several studies have investigated the homocysteine-lowering properties of pyridoxine (vitamin B6), hydroxycobalamin (vitamin B12), or folic acid alone or in combination.67891011 However, some of these studies were not placebo controlled, and therefore, they cannot distinguish the extent to which the observed effects were due to regression to the mean. Other studies were restricted to hyperhomocysteinemic subjects, healthy volunteers, or certain subgroups (eg, elderly people, men, women, or patients with cardiovascular disease or renal failure).

The aim of our study was to estimate and compare the homocysteine-lowering effect of vitamin supplementation in patients with hyperhomocysteinemia-related disease and in healthy volunteers with or without elevated homocysteine levels. Therefore, we studied the effects of an 8-week daily combined administration of 5 mg folic acid, 0.4 mg hydroxycobalamin, and 50 mg pyridoxine versus placebo on blood homocysteine levels in patients with a history of recurrent venous thrombosis and healthy volunteers. We also compared this high-dose multivitamin regimen with single-vitamin regimens of folate or hydroxycobalamin to assess which vitamin at which dose was the most effective in lowering homocysteine levels. For reasons of sample size, we restricted this “drug- and dose-finding study” to volunteers with normohomocysteinemia. Finally, we analyzed the influence of initial homocysteine and vitamin concentrations and of the 677C→T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene on the homocysteine-lowering effect of multivitamin supplementation.

Methods

The study group was recruited from subjects who had participated in a previous case-control study.4 In the current study, 92 of the 185 patients with recurrent venous thrombosis and 230 of the 500 volunteers from the general population agreed to participate in an 8-week daily vitamin supplementation trial. Six participants (three from each group) withdrew during the study. All participants were asked not to take (“self-prescribed”) vitamin supplements for at least 2 months prior to the start of the current study.

Both patients and hyperhomocysteinemic healthy volunteers were randomized to either a placebo or a high-dose multivitamin schedule. Each multivitamin tablet contained 5 mg folic acid, 0.4 mg hydroxycobalamin, and 50 mg pyridoxine. (Randomization of the volunteers had been stratified by homocysteine level in a previous study4 [cutoff point, 16 μmol/L].). Volunteers with previous homocysteine levels ≤16 μmol/L were randomized to placebo, multivitamin, or single-vitamin regimens (5 mg folic acid, 0.5 mg folic acid, or 0.4 mg hydroxycobalamin). These three additional subregimens were restricted to normocysteinemic volunteer group because the other subgroups were too small to allow randomization into more than two schedules. Randomization was performed by using the last digit of each patient’s number. So all hyperhomocysteinemic subjects and normocysteinemic patients with recurrent venous thrombosis with an odd or even number were assigned to the multivitamin or placebo group, respectively. In the normohomocysteinemic healthy volunteers, subjects with a last digit of 0 or 1 were assigned to placebo; 2 and 3, to multivitamins; 4 and 5, to 5 mg folic acid;, 6 and 7, to 0.5 mg folic acid; and 8 and 9, to 0.4 mg vitamin B12. All subjects were asked to take 1 tablet per day for 56 days. The trial was kept double-blind. Before and after the supplementation period, blood was collected after an overnight fast for homocysteine, folate, cobalamin, and pyridoxal-5′-phosphate measurements.

For homocysteine and vitamin measurements, blood samples were taken from the antecubital vein and collected into EDTA-containing tubes. Whole blood was stored at −70°C for pyridoxal-5′-phosphate determination. For the other determinations, EDTA-treated samples were immediately placed on ice and centrifuged within half an hour at 2000g for 10 minutes. The plasma was separated and stored at −20°C. The EDTA-treated samples for folate and cobalamin measurements were stored at −70°C and analyzed within 2 months. Folate and cobalamin concentrations were measured with a Dualcount SPNB (solid phase no boil) radioassay kit (Diagnostic Products Corp). Determination of pyridoxal-5′-phosphate was performed by high-performance liquid chromatography techniques according to Schrijver et al12 with some modifications.13 Total homocysteine concentrations were measured according to the method described by Fiskerstrand et al14 with some modifications.15 Mutation analysis was carried out by means of polymerase-chain reaction and restriction enzyme digestion as described elsewhere.16

In the analysis we first looked at normalization rates of homocysteine levels after multivitamin or placebo supplementation. Therefore, we calculated the fraction of hyperhomocysteinemic subjects (homocysteine >16 μmol/L in the present study) who became normohomocysteinemic (homocysteine ≤16 μmol/L) after the supplementation period. The cutoff point was a rounded value based on the 80th percentile in our previous study.4 Second, we calculated the percent homocysteine reduction for each subject and compared the median reduction in the different vitamin supplementation groups with respect to the corresponding placebo group. To compare the homocysteine-lowering effects in patients and volunteers, we later stratified the patients into a hyperhomocysteinemic and a normohomocysteinemic group according to their homocysteine levels as determined in our previous study (cutoff point, 16 μmol/L). Finally, we studied determinants of the homocysteine-lowering effect of multivitamin supplementation by calculating the median reduction in men and women; in subjects under or above 53 years of age (median age of healthy volunteers); and for several strata (tertiles) of initial homocysteine, folate, cobalamin, and pyridoxal-5′-phosphate concentrations, as well as for the three different MTHFR-genotypes (677C→T). To evaluate the difference in median reduction, we used the Mann-Whitney U test for unpaired cases (SPSS software). All participants gave their informed, written consent, and the study protocol was approved by the medical ethics committee of Leyenburg Hospital.

Results

The median age of the patient group was 62 years (range, 31 to 89) and of the volunteers, 53 years (range, 23 to 82). The median homocysteine concentration of the patient group was 13.6 μmol/L (range, 7.2 to 69) and of the volunteer group 12.8 μmol/L (range, 4.7 to 49.8).

The Figure⇓ shows the homocysteine concentrations before and after intervention for the placebo and high-dose multivitamin groups of both patients and volunteers. In the multivitamin group, 11 of 14 hyperhomocysteinemic patients with thrombosis had a normalized value after intervention (cutoff point, 16 μmol/L) compared with only 4 out 10 in the placebo group. A very similar observation was made in the healthy volunteers.

In the multivitamin group, 15 of 16 hyperhomocysteinemic subjects had a normalized value (cutoff point, 16 μmol/L) compared with only 3 of 20 in the placebo group. So for all hyperhomocysteinemic individuals together, 26 of 30 subjects had normalized homocysteine levels (<16 μmol/L) after supplementation with multivitamins compared with only 7 of 30 in the placebo group.

Table 1⇓ shows that there was no clear difference between patients with recurrent venous thrombosis and healthy volunteers with respect to their homocysteine-lowering response due to multivitamin supplementation. In both groups there was also a substantial effect in the normohomocysteinemic subjects.

Homocysteine-Lowering Effect of Multivitamin or Placebo Supplementation in Patients With Recurrent Venous Thrombosis and in Healthy Volunteers

In Table 2⇓ we compared the homocysteine-lowering effect of several single-vitamin regimens in volunteers who were normohomocysteinemic in a previous study. From these data we may conclude that the effect of 5 mg folic acid, even a low dose of 0.5 mg, is nearly as effective as the multivitamin regimen. In contrast, vitamin B12 only slightly decreased the homocysteine concentration.

Homocysteine-Lowering Effect of Several Vitamin Regimens or Placebo in Normohomocysteinemic Healthy Volunteers

In Table 3⇓ we analyzed the effects of age and sex with respect to the homocysteine-lowering effect of multivitamin supplementation. For reasons of homogeneity, we restricted this analysis to volunteers who had been randomized into either the placebo or the multivitamin group (n=120). We found a similar homocysteine-lowering effect in men and women and in subjects under and above 53 years of age.

Homocysteine-Lowering Effect of Multivitamin or Placebo Supplementation in Men and Women and in Subjects <53 or ≥53 Years Old

In Table 4⇓ we stratified the homocysteine-lowering effect on tertiles of initial homocysteine, folate, cobalamin, and pyridoxal-5′-phosphate levels. We found a stronger homocysteine-lowering effect in subjects with high initial homocysteine levels. However, even in subjects with an initial homocysteine level of <11.8 μmol/L, we still found a median reduction of 21% (range, −22 to 41%). An inverse effect was seen with respect to initial vitamin concentrations. The homocysteine-lowering effect was strongest in subjects with low folate, cobalamin, or pyridoxal-5′-phosphate concentrations.

Six of the 92 patients with recurrent venous thrombosis were homozygous for the 677C→T mutation versus 22 of the 230 control subjects (odds ratio, 0.7; 95% confidence interval, 0.3 to 1.7). In Table 4⇑ we also show that the homocysteine-lowering effect of multivitamin supplementation was not impaired in subjects homozygous for the 677C→T mutation and in fact, might even be stronger.

Discussion

We investigated the effect of multivitamin supplementation on homocysteine levels in patients with recurrent venous thrombosis and healthy volunteers from the general population. We found that combined supplementation with folic acid, hydroxycobalamin, and pyridoxine effectively reduced and normalized homocysteine levels in patients with recurrent venous thrombosis as well as in healthy volunteers. For a subgroup of normohomocysteinemic volunteers, folic acid at a dose of 5 mg or even 0.5 mg seemed to be almost as effective as the high-dose multivitamin supplementation. Oral cobalamin supplementation had only a moderate effect on homocysteine levels.

In 1985 Brattström et al6 reported a substantial homocysteine reduction in 15 volunteers who received 5 mg folic acid per day for 4 weeks. Wilcken et al7 reported a homocysteine-lowering effect of folic acid supplementation in patients with chronic renal insufficiency. Franken et al8 and van den Berg et al9 reported significant reductions in postmethionine-loading homocysteine concentrations with vitamin B6 folic acid, or a combination of both in patients with vascular disease. Although these studies were performed in large groups of patients, they were not placebo controlled and were restricted to hyperhomocysteinemic subjects, which characteristics make them rather sensitive to regression to the mean. Ubbink et al10 studied the effects of 1 mg folic acid, 0.4 mg cyanocobalamin, and 12.2 mg pyridoxal HCl alone or in combination in a placebo-controlled study in subjects with hyperhomocysteinemia. High-dose multivitamin administration resulted in a 49.8% reduction of the mean homocysteine level. Naurath et al11 studied the effect of intramuscular vitamin supplementation with folate, vitamin B6, and vitamin B12 in elderly subjects with blood vitamin concentrations in the normal range.

In our study we were able to compare the effects in patients with homocysteine-related disease (venous thrombosis) with those in healthy volunteers. These effects were quite similar. We also found about the same effects in men compared with women and in subjects under and above 53 years of age. The strongest homocysteine-lowering effect of vitamin supplementation was seen in subjects with high initial homocysteine and/or low initial vitamin concentrations. However, we also observed a moderate reduction in homocysteine levels in subjects with homocysteine and vitamin levels within the normal range. This observation raises the question of “normal” homocysteine and vitamin levels. Our definition of hyperhomocystinemia at the 80th percentile of the distribution in the general population is arbitrary. Other suggested definitions are based on mean concentrations in populations without cardiovascular disease,17 at the flat plateau of the homocysteine-folate plot.18 We think that the best definition should be based on clinical intervention studies: the lowest concentration at which vitamin supplementation reduces the risk of vascular disease. However, data on clinical studies are not yet available.

In a subgroup of normohomocysteinemic volunteers, we found approximately the same homocysteine-lowering effect of folic acid at a dose of 0.5 mg as with a dose of 5 mg. This means that considerably low doses of folic acid supplementation are effective in lowering homocysteine levels. Further studies are needed to see whether doses lower than 0.5 mg may also be effective.

Although folic acid supplementation seems to be the cornerstone in the treatment of hyperhomocysteinemia, there are some reasons for adding cobalamin and pyridoxine. First, this combination may have a stronger effect in subjects with low cobalamin or pyridoxine levels. Second, folate administration alone might mask vitamin B12 deficiency. Addition of cobalamin in sufficient dose prevents the complications of vitamin B12 deficiency, such as subacute combined degeneration of the spinal cord, even in the case of pernicious anemia.19 We did not study doses higher than 50 mg pyridoxine because higher doses may cause sensory neuropathy.2021

Recently, we found the 677C→T mutation in the MTHFR gene This mutation is associated with elevated homocysteine levels, but it is unclear whether this mutation is also associated with arterial vascular disease.24 In this study the prevalence of the 677C→T mutation in the recurrent venous thrombosis group did not really differ from that in the control group. This finding is in accordance with the results in a study of first-time venous thrombosis.25 We found that the homocysteine-lowering effect in subjects with this mutation en might be even stronger than in those without this mutation. This finding is in accordance with the study of Malinow et al,26 These results suggest that the effect of a mutated MTHFR might be “compensated” by a higher folate intake.

In conclusion, our study shows that combined supplementation with folic acid, cobalamin, and pyridoxine reduces homocysteine levels by ≈30% compared with placebo within 8 weeks in patients with recurrent venous thrombosis as well as in healthy volunteers. Whether the reduction in homocysteine levels by vitamin supplementation will lead prevention of arterial vascular disease and venous thrombosis is a major task for further clinical research.2728

Acknowledgments

This work was supported by grants from the Prevention Fund (to G.M.J.B.) (28–2263-1). We thank G.E.Th. Ferguson, pharmacist, for preparing the vitamin and placebo tablets and C. Postma, Y. Lenstra, M.T.W.B te Poele-Pothoff, A. de Graaf-Hess, D. van Oppenraaij-Emmerseel, M.F.G. Segers, L.M.F. Geelen, and J. Beunk for their excellent laboratory assistance. We wish thank R. Clarke, MD, MRCP, for his valuable comments of a previous version of the manuscript. Finally, we thank all participants for their kind contribution the study.